Trypanosoma brucei is a protozoan parasite that causes African trypanosomiasis in humans and nagana in cattle. In mammalian hosts, bloodstream form (BF) T. brucei undergoes antigenic variation and regularly switches its surface antigen, variant surface glycoprotein (VSG), to evade the host's immune attack. Although T. brucei has more than a thousand VSG genes and pseudogenes, VSG is expressed exclusively from subtelomeric loci in a strictly monoallelic manner, which ensures effectiveness of VSG switching and maximizes its efficiency. Therefore, VSG switching and monoallelic VSG expression are essential for T. brucei pathogenesis. Telomeres, being adjacent to the expression sites of VSGs, have long been proposed to play an important role in VSG expression regulation. Indeed, it has been shown that in yeast, human, and T. brucei cells, telomeres form a heterochromatic structure that affects the transcription of reporter genes targeted to subtelomeres. Particularly in yeast, this telomeric silencing has been shown to depend on telomere protein RAP1. To explore telomere functions in VSG expression and switching regulation, we have been focusing on identification of T. brucei telomere-specific proteins and characterization of their functions. We have cloned the first T. brucei telomere-binding protein, tbTRF. More importantly, we have recently identified T. brucei RAP1 in a yeast 2-hybrid screen using tbTRF as bait, confirmed that tbRAP1 is an intrinsic component of the telomere complex, and demonstrated that it is essential for silencing subtelomeric VSGs in BF T. brucei cells. This discovery reveals T. brucei telomere as a key player in surface antigen expression control and identifies tbRAP1 as a potential target for anti-parasite drugs. Our finding also shows that T. brucei is similar to a couple of other pathogens including P. falciparum and C. glabrata, in which telomeric silencing plays an important role in regulation of virulence gene expression. We plan to further study how telomere proteins, particular tbRAP1 and tbTRF, regulate VSG expression and switching. This study would be helpful for eventual eradication of T. brucei and other similar microbial pathogens. We propose several approaches to further study the functions of tbRAP1 and tbTRF. First, our observations suggest that localization of tbRAP1 to the telomere is crucial for its VSG silencing function. We therefore hypothesize that if tbRAP1 binds telomere DNA directly, this activity would be critical for VSG silencing. However, if tbRAP1 lacks any DNA binding activity, the interaction between tbRAP1 and tbTRF and the telomere binding function of tbTRF would be essential for anchoring tbRAP1 to the telomere. We will test these hypotheses in Specific Aim 1 using in vitro approaches. These studies will reveal a key mechanism for tbRAP1-mediated silencing and elucidate similar and unique features of the DNA binding and protein-protein interaction functions of tbRAP1 and other RAP1 homologs. Our work would therefore help to identify potential targets for anti-parasite drugs. In Specific Aim 2, we aim to carry out a series of in vivo genetic analyses to further understand tbRAP1's function. First, we hypothesize that tbRAP1 not only participates in VSG-silencing control but also influences VSG switching rates, which will be tested in Aim 2.1. Second, we will elucidate the relationship between different functions of tbRAP1 and to determine which domains of tbRAP1 are essential for VSG expression and/or switching regulation using systematic genetic approaches in Aim 2.2. These studies will help to reveal the underlying mechanisms of tbRAP1's function in antigenic variation. Third, we hypothesize that tbRAP1 applies its silencing effect by modulating the chromatin structure. We will therefore examine whether tbRAP1 preferentially associates with the silent chromatin and whether loss of tbRAP1 causes any changes in the chromatin structure of the derepressed ESs. Last, we hypothesize that tbRAP1 works with other unknown co-factors to establish/maintain the silencing structure at subtelomeric loci. In order to search for tbRAP1-interacting factors that also play important roles in antigenic variation, we aim to identify components of tbRAP1 protein complex by sequential immunoprecipitation. Studying functions of other factors in the same tbRAP1-mediated silencing pathway will help us to better understand the underlying mechanisms. Among the tbRAP1-interacting candidates, we may identify downstream effectors involved in VSG silencing/switching and proteins involved in the regulation of the expression, stability, or activity of tbRAP1. Identification of novel factors involved in antigenic variation also provides more potential targets for anti-parasite drugs and helps for eventual elimination of this parasite.